Cysteine oxidation in the mitogenic S100B protein leads to changes in phosphorylation by catalytic CKII-alpha subunit.
The glial-derived calcium-binding protein S100B can be secreted to act as a neurotrophic factor or a mitogen, stimulating proliferation of glial cells. The extracellular S100B activities rely on the oxidation of the protein cysteine residues (Kligman, D., and Marshak, D. R. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 7136-7139; Winningham-Major, F., Staecker, J. L., Barger, S. W., Coats, S., and Van Eldik, L. J. (1989) J. Cell Biol. 109, 3063-3071). Here we show that oxidation of the S100B cysteine residues, Cys-68 and Cys-84, induces a conformational change in the protein structure, unmasking a canonical CKII phosphorylation site located within the typical EF-hand calcium-binding site IIbeta. Intrasubunit disulfide-bridged S100B monomer and disulfide-bonded S100B dimer are phosphorylated by the catalytic CKII-alpha subunit on Ser-62 with a Km of 0.5 microM and a Vmax of 10 pmol/min/100 pmol of S100B. Oxidized S100B is the best in vitro CKII-alpha substrate identified so far. Next we show that intrasubunit disulfide-bridged S100B monomer is the most potent S100B species to stimulate [3H]thymidine uptake by C6 glial cells in culture. In addition, the phosphorylated intrasubunit disulfide-bridged S100B monomer retains apparent mitogenic activity toward C6 glial cells, and hence, 32P-labeled S100B should be a useful probe for characterizing the mechanisms by which extracellular oxidized S100B functions. Finally, we show that formation of intrasubunit disulfide-bridged S100B monomer is stimulated by peroxynitrite anion, suggesting that production of mitogenic S100B species could be enhanced in neuropathology associated with peroxynitrite anion production.
Scotto
C
C
Département de Biologie Moléculaire et Structurale du CEA, DBMS-BRCE INSERM Unité 244, Grenoble, France.
Mély
Y
Y
Ohshima
H
H
Garin
J
J
Cochet
C
C
Chambaz
E
E
Baudier
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D047390:Casein Kinase II; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D003545:Cysteine / Q000378:metabolism*; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D008934:Mitogens / Q000378:metabolism; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000737:chemistry / Q000378:metabolism*; D009457:Neuroglia; D009566:Nitrates / Q000494:pharmacology; D010084:Oxidation-Reduction; D010766:Phosphorylation; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013936:Thymidine / Q000378:metabolism
D002135:Calcium-Binding Proteins; D004220:Disulfides; D008934:Mitogens; D009414:Nerve Growth Factors; D009566:Nitrates; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; C579158:S100b protein, rat; C039642:peroxynitric acid; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D003545:Cysteine; D013936:Thymidine